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Endocrinology, doi:10.1210/en.2006-0099
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Endocrinology Vol. 147, No. 7 3219-3227
Copyright © 2006 by The Endocrine Society

Evidence for the Role of Adenosine 5'-Triphosphate-Binding Cassette (ABC)-A1 in the Externalization of Annexin 1 from Pituitary Folliculostellate Cells and ABCA1-Transfected Cell Models

Selma Omer, David Meredith, John F. Morris and Helen C. Christian

Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom

Address all correspondence and requests for reprints to: Dr. Helen C. Christian, Department of Physiology, Anatomy, and Genetics, Le Gros Clark Building, University of Oxford, South Parks Road, Oxford OX1 3QX, United Kingdom. E-mail: helen.christian{at}anat.ox.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Annexin 1 (ANXA1), a 37-kDa protein, is a member of the superfamily of Ca2+- and phospholipid-binding annexin proteins. In the anterior pituitary, ANXA1 is expressed mainly by folliculostellate (FS) cells and mediates the early delayed feedback inhibition exerted by glucocorticoids on the release of ACTH and other pituitary hormones. It has been previously demonstrated that TtT/GF cells (a FS cell line) express and externalize ANXA1 in response to glucocorticoid treatment. However, ANXA1 lacks a cleavable signal sequence and externalization is not affected by inhibitors of the secretory pathway. We have previously shown that glyburide, an ATP-binding cassette (ABC) transporter inhibitor, inhibits the externalization of ANXA1 from TtT/GF cells and pituitary tissue. Here we investigated whether ABCA1 is involved in ANXA1 externalization. The use of the ABCA1-transporter inhibitors geranyl-geranyl pyrophosphate and sulfobromophthalein significantly inhibited ANXA1 externalization. Partial silencing of ABCA1 expression in TtT/GF cells by siRNA also significantly decreased the amount of cell surface ANXA1. However, anterior pituitary tissue from ABCA1-null mice was found to externalize ANXA1 normally. Because compensation by other ABC family members may occur in vivo, ANXA1 externalization was studied in two transfection models: Xenopus oocytes injected with ABCA1 mRNA and AtT20 D1 corticoctroph cells cotransfected with ABCA1-green fluorescent protein and ANXA1. ABCA1-expressing oocytes, but not water-injected controls, were found to externalize ANXA1. Expression of ABCA1 in AtT20 D1 cells significantly increased the amount of cell surface ANXA1, compared with mock-transfected and ANXA1-only transfected controls. Together these data provide evidence for a role of ABCA1 in ANXA1 export.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NONCLASSICAL PROTEIN RELEASE independent of the endoplasmic reticulum-Golgi pathway has been reported for an increasing number of proteins lacking an N-terminal signal sequence (1). Several transport mechanisms have been demonstrated to underlie nonclassical routes of protein export from the cell. For example, IL-1{alpha} appears to be exported as a multiprotein aggregate with the Ca2+-binding protein S100A13 and copper (2), whereas transport of proteins such as IL-1ß, basic fibroblast growth factor, and macrophage migration inhibitory factor (MIF) require the function of an ATP-binding cassette (ABC) transporter (3, 4, 5). Studies investigating the secretion of annexin 1 (ANXA1), a mediator of several actions of glucocorticoid hormones, indicate that ANXA1 secretion occurs by a nonclassical secretion pathway. ANXA1 is a member of the annexin family of phospholipid- and calcium-binding proteins (reviewed in Ref. 6) and is externalized from several different cell types including macrophages, monocytes, pituitary cells, and glia (7, 8, 9, 10). Externalization of ANXA1 is stimulated by glucocorticoid treatment in a time- and concentration-dependent manner (11, 12). ANXA1 lacks a cleavable signal sequence at its N terminal (13), and its externalization is not influenced by brefeldin A, a drug that specifically inhibits estrogen receptor to Golgi transport in the classical regulated secretory pathway (14, 15).

In the anterior pituitary gland, ANXA1 plays an essential role in the manifestation of the early delayed feedback effects of glucocorticoids (for review see Ref. 16). Evidence that ANXA1 is localized mainly in the folliculostellate (FS) cells (10, 17), and is released in its phosphorylated form from FS cells in response to a glucocorticoid challenge (12, 18), have led us to propose that ANXA1 acts as a paracrine/juxtacrine mediator of glucocorticoid action in the anterior pituitary. On a temporal basis, the export of ANXA1 parallels the glucocorticoid-induced onset of the inhibition of pituitary hormone secretion, both being evident after 30 min of steroid contact and maximal at 90 min (19). Furthermore, specific high affinity proteinaceous ANXA1-binding sites are expressed on the surface of anterior pituitary endocrine cells (20). We have previously demonstrated that TtT/GF cells, a well-characterized stable mouse pituitary-derived FS cell line, express and externalize ANXA1 in response to glucocorticoid treatment specifically at the tips of the cell processes characteristic of these cells (10). The ANXA1-rich FS cells contain no classical, regulated secretory granules (10), and our studies using the ABC transport inhibitor glyburide suggest that the mechanism of externalization involves an ABC transporter (18). Furthermore, blockade of the export of ANXA1 in rat anterior pituitary with glyburide suppresses the inhibitory actions of the glucocorticoids on ACTH release (18).

The ABC transporter family constitutes a superfamily of more than 100 highly conserved proteins involved in the trans-membrane transport of a wide variety of substrates including proteins (21). ABCA1 is required for engulfment of cells undergoing apoptosis by macrophages and is involved in the translocation of phospholipids and cholesterol to apolipoprotein-AI (22, 23). Genetic deficiency of ABCA1 in humans causes Tangier disease, which is characterized by accumulation of phospholipids in the immune system (24). Because ABCA1 inhibitors such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and sulfobromophthalein (3, 5, 25) and anti-ABCA1 antisense oligonucleotides reduce IL-1ß secretion from macrophages (26), nonclassical secretion of proteins has been associated with ABCA1 protein function. Our previous studies have shown that ABCA1 is colocalized with ANXA1 surface membrane immunoreactivity in FS cells and is therefore ideally placed for a role in ANXA1 externalization (18). In addition, ABCA1 mRNA is strongly expressed in tissues that are known to export ANXA1 (lung, adrenal, pituitary, macrophages) (27). In the present study, we provide evidence that ABCA1 is involved in ANXA1 export across the plasma membrane of TtT/GF cells and in Xenopus oocyte and AtT20 D1 ABCA1 recombinant expression systems.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Drugs
The following were used for in vitro studies: glyburide, geranyl-geranyl pyrophosphate (GGPP), sulfobromophthalein (BSP), and dexamethasone sodium phosphate. GGPP and BSP are ABCA1 transporter inhibitors (25). Glyburide was dissolved initially in a small amount of dimethylsulfoxide and subsequently diluted in incubation medium; the final concentration of dimethylsulfoxide never exceeded 0.01% and appropriate controls were included in all experiments. Dexamethasone was initially dissolved in a small amount of ethanol and subsequently diluted with incubation medium immediately before use; the final concentration of ethanol did not exceed 0.1%. GGPP and BSP were dissolved and diluted in incubation medium. All drugs and reagents were from Sigma Chemical Co. (Poole, Dorset, UK) unless otherwise stated.

Animals
ABCA1-null and wild-type mice (22), with a DBA/1 background, were generously supplied by Dr. Giovanna Chimini (Centre d’Immunologie, Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique de Marseille Luminy, Marseille, France). Male wild-type littermate control and ABCA1-null mice (5–6 months of age) were maintained on a standard chow pellet diet with tap water ad libitum. Animals were housed in groups of five per cage in a quiet room with controlled lighting (lights on 0800–2000 h) and temperature maintained at 21–22 C. All experiments were started between 0800 and 0900 h to avoid changes associated with the circadian rhythm. The Principles of Laboratory Animal Care (National Institutes of Health publication no. 85–23) were followed, and animal work was carried out under license in accordance with the U.K. Guidance on the Operation of Animals, Scientific Procedures Act 1986. Mice were killed by stunning followed by decapitation, the pituitary gland removed, and the anterior lobe separated from the posterior lobe.

Mature large female X. laevis were obtained from Blades Biological (Kent, UK).

Cell lines
Murine FS-like (TtT/GF) and corticotroph (AtT20 D1) cell lines were maintained as previously described (28) in DMEM-Ham’s F12 medium (Invitrogen Ltd., Paisley, UK) enriched with 15% (vol/vol) fetal calf serum (FCS; PAA Laboratories Ltd., Yeovil, UK), penicillin (100 IU/ml), and streptomycin (100 µg/ml) in a 5% CO2 humidified atmosphere.

TtT/GF cell incubations
TtT/GF cells were plated at 50,000 cells/well in 24-well plates in DMEM and 1% FCS and allowed to reach 80% confluence before treatments. They were then incubated for 3 h either in DMEM and 1% FCS alone or with glyburide (100 µM), GGPP (10 µM), or BSP (100 µM) with or without 0.1 µM dexamethasone sodium phosphate. At the end of the incubation period, cells were processed for Western blot analysis of cell surface ANXA1 expression.

Externalization of ANXA1 in vitro by anterior pituitary segments
Anterior pituitary glands from wild-type and ABCA1 null mice were cut into two roughly equal segments. The segments were distributed randomly (one segment per well) in the wells of 24-well tissue culture plates and incubated at 37 C for 3 h in 1 ml DMEM containing 0.1 µM dexamethasone sodium phosphate under a humidified atmosphere saturated with 95% O2-5% CO2. The medium was changed after 1 and 1.5 h. At the end of the incubation period, segments were processed for subsequent Western blot analysis of cell surface ANXA1 expression.

Silencing of ABCA1 expression in TtT/GF cells
Small interference (si) RNA synthesis and transfection.
For ABCA1 RNA inhibition, four siRNA duplexes were designed from the full-length mouse ABCA1 sequence (siRNA1, 5'-AAGACCACCACCATGTCAATA-3'; siRNA2, 5'-AAGCCCTCTTTGGAGGG AATA-3'; siRNA3, 5'-AACAGGTTTGGAGATGGTTAT-3'; siRNA4, 5'-AACGGGTGTCTAC GTGCAACA-3', corresponding to positions 2815–2835, 992-1012, 6397–6417, and 1842–1862 of GenBank accession no. NM-01345, respectively). Each targeting segment was searched with National Center for Biotechnology Information blast to confirm specificity only to the targeted gene. The siRNAs were synthesized commercially by QIAGEN (Crawley, UK; high-performance purity grade), and each siRNA duplex underwent stringent quality control including matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. TtT/GF cells were maintained in six-well plates at 60–80% confluence. The siRNA transfection was carried out with RNAiFect transfection reagent (QIAGEN) according to the manufacturer’s protocol. Three days after transfection with the siRNA duplexes, the cells were subjected to real-time PCR assay of ABCA1 expression.

Real–time PCR.
The transfected cells were harvested, and the silencing effect was monitored by real-time PCR. Total RNA was isolated using TRI reagent and treated with DNase (DNA-free; both Ambion, Witney, UK) to remove traces of genomic DNA contamination. Five micrograms of total RNA from each sample was subsequently reverse transcribed using 1 µl Moloney murine leukemia virus reverse transcriptase (CLONTECH, Oxford, UK), 2 µl 10 mM deoxynucleotide triphosphate, 2 µl 100 mM dithiothreitol, in a total volume of 20 µl. ABCA1 expression was then detected and quantified by real-time PCR using 12.5 µl of Absolute QPCR mix (ABgene, Epsom, UK), 0.4 µM of each of ABCA1 forward primer (5'-CAGCTTGGTGATGCGGAAGT-3') and ABCA1 reverse primer (5'-CTCGGCTGCATCGAGCTT-3') and 0.25 µM ABCA1, FAM/TAMRA-dual-labeled probe (5'-TCTGCCCCTCTGTGGTCTACCGAGGAA-3') (Sigma-Genosys, Hinxton, UK) designed from the mouse ABCA1 sequence in a total volume of 25 µl. The QPCR assay was performed in an ABI PRISM 7000 (Applied Biosystems, Warrington, UK) and cycled as follows: initial enzyme activation at 95 C for 15 min and then 40 cycles of 95 C for 15 sec and 60 C for 1 min. The assays were normalized with 18S RNA primers and probes (Applied Biosystems) and the results expressed as the amount of ABCA1 mRNA/18S RNA.

Expression of ABCA1 in Xenopus oocytes
In vitro transcription of ABCA1 construct.
The full-length mouse ABCA1 cDNA sequence, subcloned into pSP64TN poly A vector (29), was used to generate cRNA for oocyte expression. The construct was linearized and purified using the PCR purification kit (QIAGEN), and cRNA generated by in vitro transcription using SP6 DNA polymerase (Roche Diagnostics, Lewes, UK) for 1 h at 37 C. The resultant cRNA was DNase treated and purified using the RNeasy minikit (QIAGEN) following the manufacturer’s protocol. The final cRNA concentration was determined by spectrophotometric reading at 260 nm and formaldehyde-agarose gel analysis and adjusted to 1 µg/ml.

Oocyte preparation.
X. laevis oocytes (stage V and VI) were obtained by ovariectomy under MS-222 anesthesia in accordance with U.K. Home Office regulations as previously described (30) and maintained at 18 C in modified Barth’s medium [88 mM NaCl, 1 mM KCl, 0.42 mM MgSO4, 0.84 mM NaHCO3, 0.82 mM CaCl2, 5 mM HEPES, 5 mM sodium pyruvate, 50 µg/ml gentamicin (Fluka, Poole, UK) (pH 7.6) with NaOH]. Experiments were performed at least 72 h after microinjection of oocytes with either 50 ng of ABCA1 cRNA or 50 nl water (negative control). For determination of ABCA1 protein expression, oocytes were homogenized in lysis buffer (10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100 in PBS) and immunoprecipitated using 5 µl of rabbit anti-ABCA1 antibody (Novus Biochemicals, Littleton, CO), and protein A Sepharose followed by SDS-PAGE and Western blot analysis for ABCA1 as previously described (18). The oocytes were processed for Western blot analysis of surface and intracellular ANXA1 as described below.

Coexpression of ANXA1 and ABCA1 in AtT20 D1 cells
A bidirectional pBI-ABCA1/green fluorescent protein (GFP) vector encoding a full-length GFP-tagged ABCA1 fusion protein (22) was used for mammalian expression of ABCA1 in AtT20 D1 cells. The full-lengthANXA1 was amplified from mouse pituitary cDNA using specific primers that spanned the full-coding sequence containing restriction sites (shown in bold) for unidirectional cloning: ANXA1 forward, 5'-AGCTCAGCTAGCAGATGGCAATGGTATCAGAATTC-3'; ANXA1 reverse, 5'-AGCTCAAAGCTTGATGTCTAGTTTCCACCAC-3'. The PCR product was then cloned into pcDNA3.1 vector (Invitrogen) following NheI and HindIII restriction digestion. Cells were either cotransfected for 24 h with the ABCA1-GFP and ANXA1 constructs, singly transfected with the ANXA1 construct or mock transfected using effectine transfection reagent (QIAGEN) following the manufacturer’s protocol. The transfection complexes were removed 10 h after transfection, the cells washed with PBS, and subsequently grown for 24 h. At the end of the incubation, the cells were processed for immunofluorescence and fluorescence-activated cell sorter (FACS) analysis of cell surface ANXA1 expression.

Detection of ANXA1 by SDS-PAGE and Western blot analysis
ANXA1 was extracted from pituitary tissue, TtT/GF cells, and Xenopus oocytes as described previously (18). Briefly, cell surface ANXA1 was removed from the outer cell membranes by washing the tissue or cells gently for 2 min in a solution containing 1 mM EDTA in PBS, which, by chelating Ca2+, releases ANXA1 into the medium from Ca2+-dependent cell surface binding sites. Intracellular ANXA1 was extracted from the remaining tissue by sonication (25 Hz, 20 sec, Soniprep 150; MSE, London, UK) on ice in EDTA (10 mM) containing Triton X-100 (1% vol/vol) in PBS. The protein content of each sample was determined using the BCA protein assay (Pierce, Cheshire, UK). The samples were then analyzed for ANXA1 by SDS-PAGE and subsequent Western blotting using a well-characterized polyclonal anti-ANXA1 antibody (anti-ANXA1 pAb, raised in sheep against the full-length human recombinant ANXA1 and diluted 1:10,000) (16) as a probe. The housekeeping protein {alpha}-tubulin was also detected with a mouse anti-{alpha}-tubulin antibody (clone B-5-1-2; 1:5000 1 h, room temperature) and mouse secondary antibody (1:5000, 1 h, room temperature; both Sigma). On no occasion was {alpha}-tubulin detected in cell surface EDTA washes, confirming the absence of cellular material in the washes. Immunoreactive protein bands were detected by chemiluminescence using enhanced chemiluminescence reagents and exposed to Hyperfilm (both from Pierce). The blots were scanned using a flatbed scanner [HP (Uxbridge, UK) Scanjet 5200 with Adobe (Bracknell, UK) Photodeluxe Business Edition, version 1.1] and the band intensity analyzed using the TINA software program (TINA, version 2.10, Raytest; Isotopenmessgeraete GmbH, Germany). Intensity values were normalized relative to control values.

Quantification of cell surface ANXA1 expression by FACS analysis
Aliquots of the cell suspension (2 x 105 cells in 100 µl) were added to a 96-flat well plate in triplicate and incubated at 4 C in the presence or absence (controls) of anti-ANXA1 mouse monoclonal antibody (Biogen Research Corp., Cambridge, MA; 1:500) diluted in buffer A [25 mM HEPES, 1 mM CaCl2, 2% (wt/vol) BSA, 1 mM Mg Cl2]. The cells were then washed in cold buffer A and incubated on ice for 30 min with F(ab')2 fragments of goat antimouse IgG fluorescein isothiocyanate-conjugated secondary antibody (diluted 1:100 in buffer A; Caltag, San Francisco, CA). After further washing in buffer A, the cell surface fluorescence was analyzed by flow cytometry. In all cases a Becton Dickinson FACScan II analyzer with air-cooled 100-mW argon ion laser and Consort 32 computer running Lysis II software (Becton Dickinson and Co., Oxford, UK) was used. At least 10,000 cells/sample were counted and characterized by dot plot according to their forward and side scatter characteristics. The data were analyzed as units of fluorescence measured in the FL1 channel (mean fluorescence intensity). Within each experiment the data were normalized to background (i.e. fluorescence due to second antibody alone).

Detection of cell surface and intracellular ANXA1 in transfected AtT20 D1 cells by immunofluorescence microscopy
AtT20 D1 cells, plated onto 8-well chamber slides (LAB-TEK, Nalge Nunc International, Hereford, UK) were cotransfected with ABCA1 and ANXA1 following the methods above. The cells were fixed in freshly prepared 3% formaldehyde and 0.05% glutaraldehyde in PBS for 5 min. For visualization of intracellular ANXA1 immunoreactivity, cells were permeabilized with 0.2% Triton X-100 (in PBS at room temperature) for 5 min; this step was omitted for detection of surface ANXA1 immunoreactivity. Nonspecific antibody binding sites were blocked with block buffer (3% BSA in PBS; PBS-BSA) for 30 min. Slides were incubated overnight at 4 C with sheep antihuman ANXA1 antibody (1:1000) diluted in PBS-BSA, washed with PBS-BSA, and then incubated for 1 h at room temperature with a Texas red-conjugated donkey antisheep secondary antibody (1:100; Vector Laboratories, Burlingame, CA). The culture chambers were removed and the slides mounted in Vectashield mounting medium (Vector Laboratories) and examined using a TCS confocal microscope (Leica Corp. Microsystems, Wetzlar GmbH, Germany). Nonspecific staining and background were assessed by substitution of nonimmune sheep sera for primary antisera and incubation with second antibodies alone.

Detection of ANXA1 in transfected AtT20 D1 cells by immunogold electron microscopy
For immunoelectron microscopy, sucrose-cryoprotected mock-transfected, ANXA1-transfected, and ABCA1 and ANXA1 cotransfected AtT20 D1 cells were slam frozen, freeze substituted at –80 C in methanol for 48 h and embedded at –20 C in LR Gold acrylic resin (Agar Scientific, Essex, UK). Ultrathin sections (50–80 nm) were prepared by use of a Reichert Ultracut S microtome and mounted on 200-mesh nickel grids. Immunogold labeling for ANXA1 was performed as previously described (10). Sections were incubated with sheep antihuman ANXA1 polyclonal antibody at a dilution of 1:1000 for 2 h and 1 h with antisheep IgG 15 nm gold complex (British Biocell, Cardiff, UK). For control sections, the primary antibody was omitted and replaced with 0.1 M phosphate buffer containing 1% (wt/vol) egg albumin. Sections were then lightly counterstained with uranyl acetate and lead citrate. All antibodies were diluted in 0.1 M phosphate buffer containing 1% (wt/vol) egg albumin. The sections were viewed with a JEOL 1010 transmission electron microscope (JEOL, Peabody, MA).

Data analysis
Semiquantitative measures of ANXA1 expression were made by comparisons of Western blot band ODs (arbitrary units) to give a relative numerical guide to the ratios of the band intensities and their variance. Responses to dexamethasone were calculated as a percentage of the corresponding drug-free control and expressed as the mean ± SEM (n = 3 gels); statistical comparisons between the normally distributed data from groups were made by the standard t test. Each of the studies was repeated three times and in all instances the data profile was similar. The data derived by FACS analysis were normally distributed (Shapiro and Wilkes test) and were analyzed by standard parametric tests and ANOVA with post hoc comparisons by the Bonferroni test. Statistical comparisons were made on the mean data from three experiments (n = 3), each comprising three cell aliquots per experimental group. The data are expressed as the mean ± SEM. Differences were considered significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of GGPP and BSP on externalization of ANXA1 by TtT/GF cells
Initial studies confirmed that ANXA1 was readily detectable in the TtT/GF cells by Western blot analysis. The major band corresponded to the native biologically active 37-kDa species of the protein. A second band of approximately 32 kDa (which is reported to represent a metabolite) (8) was also detected. Figure 1Go demonstrates the effects of glyburide (100 µM), GGPP (10 µM), and BSP (100 µM) on the amount of ANXA1 on dexamethasone (0.1 µM)-induced externalization of ANXA1. A typical Western blot is shown in Fig. 1AGo. The inhibitor concentrations used have been reported in the literature to produce optimal inhibition of IL-1ß, IL-12, and MIF secretion from a variety of cell types (3, 5, 25, 31). As previously reported (18), glyburide treatment for 3 h in culture alone did not affect the amount of ANXA1 detected on the surface of TtT/GF cells [Fig. 1AGo (glyburide treated, lanes 5 and 6 vs. control lanes 1 and 2) and Fig. 1BGo]. Cotreatment of TtT/GF cells with glyburide (3 h) blocked (P < 0.01) the increase in surface ANXA1 induced by dexamethasone (lanes 7 and 8, dexamethasone and glyburide vs. lanes 3 and 4, dexamethasone alone; Fig. 1Go, A and B). Similarly neither BSP (BSP treated, lanes 9 and 10 vs. control lanes1 and 2; Fig 1Go, A and B) nor GGPP treatment for 3 h (GGPP-treated, lanes 13 and 14 vs. control lanes 1 and 2; Fig 1AGo and B) affected the amount of ANXA1 detected on the surface of TtT/GF cells. However, both BSP and GGPP blocked (P < 0.01) the dexamethasone-induced translocation of ANXA1 (lanes 11 and 12, dexamethasone and BSP, and 15 and 16, dexamethasone and GGPP, vs. lanes 3 and 4, dexamethasone alone; Fig 1Go, A and B).


Figure 1
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  		FIG. 1. Effects of glyburide, BSP, and GGPP on control (untreated) and dexamethasone-stimulated ANXA1 externalization by TtT/GF cells. Surface ANXA1 (EDTA wash) was measured by Western blot analysis. A, Western blot: control, lanes 1 and 2; 0.1 µM dexamethasone treated, lanes 3 and 4; 100 µM glyburide treated, lanes 5and 6; dexamethasone and glyburide treated, lanes 7 and 8; 100 µM BSP treated, lanes 9 and 10; BSP and dexamethasone treated, lanes 11 and 12; 10 µM GGPP treated, lanes 13 and 14; GGPP and dexamethasone treated, lanes 15 and 16. B, Integrated densitometry data. Open columns, Control; hashed columns, dexamethasone treated. Values expressed as mean ± SEM, n = 3 experiments; **, P < 0.01 vs. negative control; 2+, P < 0.05 vs. dexamethasone-alone group, standard t test.

 
Externalization of ANXA1 from ABCA1 null mouse anterior pituitary and ABCA1 partially silenced TtTGF cells
Figure 2Go illustrates the amount of ANXA1 in wild-type and ABCA1 null anterior pituitary cells on the outer surface of the cells (i.e. in EDTA washes) and in the remaining tissue extract representing the intracellular ANXA1. No significant difference was detected in the amount of cell surface ANXA1 on anterior pituitary cells from wild-type and ABCA1 null mice (Fig. 2Go, A and D). However, a significantly greater amount of ANXA1 (P < 0.05) was detected intracellularly in ABCA1 null anterior pituitary, compared with wild type (Fig. 2Go, B and D), whereas no change was observed in the amount of intracellular {alpha}-tubulin detected (Fig. 2CGo).


Figure 2
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  		FIG. 2. Western blot and quantitation showing the amount of ANXA1 in the EDTA wash, i.e. cell surface ANXA1 (A); intracellular ANXA1 (B); and intracellular {alpha}-tubulin in wild-type and ABCA1-null anterior pituitary tissue (C). Wild-type, lanes 1 and 2; ABCA1-null, lanes 3 and 4. D, Histograms showing integrated densitometry data. Open columns, wild-type tissue, hashed columns, ABCA1-null tissue. Values expressed as mean ± SEM, n = 3 experiments; *, P < 0.05 vs. wild-type, standard t test.

 
Real-time PCR assay of ABCA1 mRNA expression in TtT/GF cells transfected with each of the custom-designed siRNA duplexes revealed that duplex siRNA3 produced the greatest silencing effect (Fig 3AGo). A 60% reduction in ABCA1 mRNA expression was measured in siRNA3 silenced cells, compared with mock-transfected cells (Fig 3AGo; normalized to 18S RNA mRNA expression), and therefore, siRNA3 was used in subsequent experiments. Figure 3BGo illustrates the amount of cell surface ANXA1 detected by FACS analysis on mock-transfected control and siRNA3 ABCA1 transfected TtT/GF cells. A significant decrease (P < 0.05) in surface ANXA1 was measured in siRNA3 silenced cells, compared with mock-transfected cells (Fig 3BGo).


Figure 3
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  		FIG. 3. Effect of partial silencing of ABCA1 in TtT/GF cells on the expression of cell surface ANXA1 measured by FACS analysis. A, Histograms showing the ABCA1 gene knockout achieved with siRNA1, 2, 3, and 4 normalized to 18s RNA mRNA expression. B, Mean data (expressed as mean of intensity of fluorescence, FL1) of cells stained for cell surface ANXA1 in control, mock-transfected, and siRNA3 silenced TtT/GF cells from three experiments. Values expressed as mean ± SEM, n = 3; P < 0.05 vs. control, ANOVA and Bonferroni test.

 
Expression of ABCA1 in Xenopus oocytes and measurement of cell surface ANXA1
Initial studies demonstrated that ANXA1 was readily detectable in Xenopus oocytes by Western blot analysis. A single band was detected which corresponded to the biologically active 37-kDa molecular mass species of the protein. After injection of cRNA, the synthesis and overexpression of ABCA1 at the predicted size (220 kDa) was detected by immunoprecipitation and Western blotting (Fig. 4AGo, lane 2 vs. water-injected control lane 1). Surface and intracellular ANXA1 was detected by Western blot analysis in water-injected and ABCA1-expressing oocytes. In water-injected oocytes, all the ANXA1 protein was detected in the intracellular lysates (Fig. 4DGo, lanes 1 and 2), and ANXA1 was not detected in the surface ANXA1 EDTA wash (Fig. 4BGo, lanes 1 and 2). However, expression of ABCA1 substantially increased ANXA1 expression in the surface ANXA1 EDTA wash (Fig. 4BGo, lanes 3 and 4) but did not apparently affect intracellular ANXA1 content (Fig. 4DGo, lanes 3 and 4), although expression levels were so high as to saturate the detection system. No change was observed in the amount of {alpha}-tubulin detected in intracellular extracts (Fig. 4EGo), and no {alpha}-tubulin immunoreactive bands were detected in cell surface EDTA washes (Fig. 4CGo), confirming oocyte membrane integrity.


Figure 4
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  		FIG. 4. Western blots showing expression of ANXA1 in control, water-injected, and ABCA1-GFP mRNA-injected Xenopus oocytes. A, Western blot showing ABCA1 expression in control water-injected and ABCA1-transfected oocytes. Control water-injected oocytes, lane 1; ABCA1 mRNA injected, lane 2. B, EDTA wash of cell surface ANXA1. C, EDTA wash of {alpha}-tubulin. D, Intracellular ANXA1. E, Intracellular {alpha}-tubulin. B–E, Control water-injected oocytes are shown in lanes 1 and 2 and ABCA1 cRNA-injected oocytes in lanes 3 and 4.

 
Expression of ANXA1 and ABCA1-GFP in AtT20 D1 cells and determination of surface ANXA1
Immunofluorescence microscopy (Fig. 5Go), immunogold electron microscopy (Fig. 6Go), and FACS analysis (Fig. 7Go) confirmed expression of ANXA1 in AtT20 D1 cells cotransfected with ANXA1 and ABCA1. In fixed and permeabilized AtT20 D1 cells, strong immunofluorescent staining for ANXA1 extended throughout the cytoplasm (Fig. 5Go, A, C, and E). In AtT20 D1 cells that had been fixed but not permeabilized, ANXA1 was concentrated in several peripheral patches of immunoreactivity (Fig. 5BGo). ABCA1-GFP fluorescence was similarly distributed (Fig. 5Go, D and F). Figure 5GGo illustrates the absence of ANXA1 immunofluorescence, and Fig. 5HGo the absence of green fluorescence, in fixed and permeabilized mock-transfected cells. No immunolabeling was detected in nonimmune serum or secondary antibodies-only controls (data not shown). Immunogold localization of ANXA1 confirmed diffuse ANXA1 localization over the cytoplasm of the AtT20 D1 cells transfected with ANXA1 (Fig. 6BGo ANXA1 alone, Fig. 6CGo transfected with ANXA1 and ABCA1-GFP) but was absent in mock-transfected controls (Fig. 6AGo). ANXA1 immunoreactivity was absent at the plasma membrane in ANXA1-only transfected cells but was visualized in ANXA1 and ABCA1-cotransfected cells (Fig. 6CGo vs. Fig. 6BGo). In agreement with these observations, quantitation of cell surface ANXA1 by FACS analysis (Fig. 7Go) demonstrated a significant (P < 0.05) increase in surface ANXA1 detected on ANXA1- and ABCA1-cotransfected cells, compared with ANXA1-only transfected and mock-transfected cells.


Figure 5
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  		FIG. 5. Immunofluorescence confocal microscopy showing expression of ABCA1-GFP and surface ANXA1 in ANXA1 and ABCA1-GFP cotransfected AtT20 D1 cells. A, C, and E, Fluorescence images of immunofluorescent labeling in fixed and permeabilized conditions. B, D, and F, Fluorescence images of labeling in fixed but not permeabilized conditions. In permeabilized conditions, ANXA1 is revealed in the cytoplasm and bright patches of external ANXA1. A and B, ANXA1. C and D, ABCA1-GFP. E and F, overlay ANXA1 and ABCA1-GFP. Arrows indicate bright patches of external ANXA1 and/or ABCA1. Absence of ANXA1 immunofluorescence (G) and absence of green fluorescence (H), in fixed and permeabilized mock-transfected cells. No immunofluorescence was detected in nonimmune serum or secondary antibodies only controls (data not shown). Magnification, x500.

 

Figure 6
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  		FIG. 6. Electron micrographs of AtT20 D1 cells showing immunogold detection of ANXA1. Nontransfected (A), ANXA1-transfected (B), and ANXA1- and ABCA1-cotransfected (C) cells. Arrows indicate ANXA1 immunogold particles. Scale bars, 300 nm.

 

Figure 7
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  		FIG. 7. FACS analysis of the amount of cell surface ANXA1 expression in control nontransfected, ANXA1-transfected, and ABCA1-transfected AtT20 D1 cells. Mean data (expressed as mean of intensity of fluorescence, FL1) from three experiments. Values expressed as mean ± SEM, n = 3; P < 0.05 vs. control, ANOVA and Bonferroni test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study provides evidence for a role of the ABC transporter ABCA1 in the mechanism of secretion of ANXA1. ANXA1 export by anterior pituitary tissue is well established despite the fact that ANXA1 lacks the hydrophobic signal sequence, which targets proteins to the classical secretory pathway (13). Our previous studies using the ABC transport inhibitor glyburide strongly suggested that ANXA1 externalization is mediated by an ABC transporter mechanism (18). Among the several subclasses of ABC transporters, ABCA1 was selected as a candidate to investigate as a putative ANXA1 transporter because it has been shown to mediate translocation of several proteins lacking a signal sequence (1). Consistent with this hypothesis, our RT-PCR and Western blot studies demonstrated that TtTGF cells, enriched primary FS cells, and mouse anterior pituitary express ABCA1 (18). Furthermore, immunofluorescence labeling in TtT/GF cells showed that ABCA1 immunoreactivity was colocalized with externalized ANXA1 in specific foci on the plasma membrane (18).

Because glyburide inhibits other ABC transporters in addition to ABCA1 (e.g. sulfonylurea receptors) and because it is likely that other ABC transporter isoforms are expressed in FS cells, we initially examined whether other ABCA1 inhibitors can mimic the effect of glyburide. We tested the ABCA1 inhibitors BSP (25, 29) and GGPP (31). GGPP, one of the major products of the mevalonate pathway, acts by potently suppressing ABCA1 expression (30). It should be noted that BSP is not a truly selective inhibitor of ABCA1 because it also inhibits the organic anion transporter, organic anion transporting polypeptide-1 (32). Both drugs inhibited ANXA1 externalization, further supporting the view that ABCA1 is involved in ANXA1 export. To test this hypothesis further, we investigated dexamethasone-stimulated externalization of ANXA1 in anterior pituitary tissue from ABCA1-null mice (22). Intracellular ANXA1 was significantly increased in the ABCA1-null mice, raising the possibility that export of ANXA1 had been reduced, resulting in accumulation of intracellular stores. However, no difference in the amount of ANXA1 at the cell surface was detectable in ABCA1 null mice, compared with wild type. The reason for these apparently contradictory findings is not clear, although what determines how much and how long AnxA1 remains bound to the cell membrane is not known.

Compensation by other ABC family members may occur in vivo in the ABCA1-null mouse. We therefore used a siRNA-mediated gene silencing approach to evaluate the role of ABCA1 in ANXA1 transport. Four different siRNAs designed to target ABCA1 were each transfected into TtT/GF cells. Quantitative real-time PCR revealed that one of the siRNAs, siRNA3, was able to reduce endogenous ABCA1 expression by 60%. Although complete ablation of ABCA1 was not achieved, we found that knocking down ABCA1 with siRNA3 significantly decreased the amount of surface ANXA1 immunoreactivity detected by flow cytometric analysis, compared with mock-transfected TtT/GF cells.

Our ABCA1 transfection studies in Xenopus oocytes injected with ABCA1 cRNA and AtT20 D1 corticotroph cells cotransfected with ABCA1-GFP and ANXA1 add further support to the hypothesis that ABCA1 plays a role in ANXA1 transport. Xenopus oocytes were found to express abundant endogenous ANXA1 and a low, but detectable, amount of ABCA1 but did not externalize ANXA1. A significant increase in surface ANXA1 was detected in oocytes overexpressing recombinant ABCA1 but not in water-injected controls. In accordance with our previous Western blot and RT-PCR analysis (28), we were unable to detect ANXA1 protein in the AtT20 D1 cells by immunogold electron or confocal immunofluorescence microscopy. However, AtT20 D1 cells do express ANXA1-binding sites (28), as do primary corticotrophs and other pituitary endocrine cells (20). Visualization by confocal immunofluorescence microscopy of ANXA1 associated with the surface membrane of ANXA1-ABCA1-GFP transfected AtT20 D1 cells showed that ANXA1 appears as punctuate foci of immunoreactivity. This finding was not unexpected because, in FS cells (10) and other well-studied ANXA1-externalizing cells, surface endogenous ANXA1 immunoreactivity also appears to be localized to patches on the surface of the cells, i.e. in A549 lung adenocarcinoma cells (17) and human neutrophils (33). In contrast, although ANXA1-only transfected cells contained abundant intracellular ANXA1, surface ANXA1 immunoreactivity was not detectable by FACS analysis or immunogold electron microscopy. However, within ANXA1-only and ANXA1-ABCA1-GFP-cotransfected AtT20 D1 cells, immunogold electron microscopy confirmed that the exogenous recombinant intracellular ANXA1 protein expressed was not packed or concentrated into any detectable form of subcellular organelle but was free within the cytosol as in pituitary FS cells (10). Therefore, it would appear that the transfected exogenous ANXA1 is targeted to its correct intracellular locations but is not able to access extracellular sites in the absence of ABCA1 expression.

In conclusion, these studies provide evidence that ANXA1 is secreted by a nonclassical pathway dependent on ABCA1 transporter function. We do not exclude, however, that other nonclassical mechanisms may contribute to ANXA1 release. For example, nonclassical secretion of a closely related annexin family member, annexin 2, has been shown to be dependent on S100A10 protein expression in human umbilical vein endothelial cells (34). S100 is a marker protein for FS cells, but the specific isoforms expressed and their function remain unclear (35). Furthermore, it is likely that ABCA1 is involved in the nonclassical release of other paracrine messengers that are released by FS cells, such as basic fibroblast growth factor (36) and MIF. MIF, a proinflammatory cytokine previously shown to require ABCA1 for its release from the THP-1 human acute monocytic leukemia cell line (5), has recently been shown to be expressed by FS cells (37).


   Acknowledgments
 
We thank Dr. Giovanna Chimini (Marseille, France) for the ABCA1-null mice, Lynne Scott and Sarah Rodgers for expert technical support, and Dr. Paola Dalton (Department of Physiology, Anatomy, and Genetics, University of Oxford) and Dr. Jo Payne (Hammersmith Hospital, Imperial College, London, UK) for invaluable help with FACS analysis.


   Footnotes
 
Research in the authors’ laboratory was supported by the Wellcome Trust.

S.O., D.M., J.F.M., and H.C.C. have nothing to declare.

First Published Online April 6, 2006

Abbreviations: ABC, ATP-binding cassette; ANXA1, annexin 1; BSP, sulfobromophthalein; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; FS, folliculostellate; GFP, green fluorescent protein; GGPP, geranyl-geranyl pyrophosphate; MIF, migration inhibitory factor; si, small interference.

Received January 24, 2006.

Accepted for publication March 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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