Previous results indicate that apoE (apolipoprotein E) may be associated with the nucleus in specific cell types, particularly under stress conditions such as serum starvation. In addition, nuclear apoE localization in ovarian cancer was recently shown to be correlated with patient survival. In order to better understand the factors associated with apoE nuclear localization, we examined intracellular apoE trafficking using live-cell imaging of CHO (Chinese-hamster ovary) cells that constitutively expressed apoE–GFP (green fluorescent protein). In addition, we used biotinylated apoE (in a lipid-free state and as a lipidated discoidal complex) to track the uptake and potential nuclear targeting of exogenous apoE. Our results indicate that a small proportion of apoE–GFP is detected in the nucleus of living apoE–GFP-expressing CHO cells and that the level of apoE–GFP in the nucleus is increased with serum starvation. Exposure of control CHO cells to exogenous apoE–GFP did not result in nuclear apoE–GFP localization in the recipient cells. Similarly, biotinylated apoE did not reach the nucleus of control CHO cells or SK-N-SH neurons. In contrast, when biotinylated apoE was delivered to recipient cells as a lipidated apoE disc, apoE was detected in the nucleus, suggesting that the lipoprotein complex alters the intracellular degradation or trafficking of apoE. Biotinylated apoE discs containing each of the three common human apoE isoforms (E2, E3 and E4) were also tested for nuclear trafficking. All three apoE isoforms were equally detected in the nucleus. These studies provide new evidence that apoE may be targeted to the nucleus and shed light on factors that regulate this process.
- apolipoprotein E (apoE)
- green fluorescent protein (GFP)
- live-cell imaging
- nuclear targeting
ApoE (apolipoprotein E) is a ∼34 kDa glycoprotein that plays an important role in plasma lipoprotein metabolism and cellular lipid transport . ApoE facilitates endocytosis of lipoproteins via the LDLr (low-density lipoprotein receptor) and related LDLr family members and, in humans, there are three major apoE isoforms, E2, E3 and E4, which differ in their cysteine/arginine residue composition at positions 112 and 158 [2,3]. The apoE2 isoform (when associated with type III hyperlipoproteinaemia) and the apoE4 isoform are associated with increased atherosclerosis risk [3,4]. The apoE4 isoform is a risk factor for late-onset Alzheimer's disease [5,6]. The complete lack of apoE expression in ApoE gene knockout (ApoE−/−) mice results in severe atherosclerosis and neurological abnormalities, including memory and learning defects [7,8].
ApoE is constitutively expressed at high levels in macrophages, hepatocytes and astrocytes, and is detectable in most peripheral tissues . ApoE expression may also be induced under certain types of cellular stress. For example, apoE expression is transcriptionally up-regulated during apoptosis in neurons and fibroblasts [9,10]. Serum starvation also induces apoE expression in fibroblasts and astrocytoma cell lines [9,11]. Other studies indicate that brain apoE expression may be increased as a consequence of aging  and in specific types of cancer [13,14]. The role that apoE plays under these various inducible/stress conditions is unclear; however, in the case of apoptosis, apoE appears to facilitate clearance of apoptotic debris which would provide a pathway for re-utilization of membrane lipids [10,15]. ApoE also has additional biological functions not directly related to lipid transport and these include roles in antioxidant actions, regulation of cell signalling, immunoregulation and in cancer [1,16–18].
An intriguing and largely unexplored aspect of cellular apoE expression and function is the finding that apoE has also been detected in the nucleus. Earlier studies identified apoE in a nuclear fraction of rat liver, and our group subsequently detected small amounts of apoE associated with the nucleus of human fibroblasts [9,19]. More recently, apoE was detected in the nucleus of ovarian cancer cells . Of potential importance in the studies by Chen et al.  is the fact that apoE in the nucleus appeared to be associated with better survival in ovarian cancer patients. In our own studies, we have found that serum starvation can lead to increased levels of nuclear apoE; however, since total apoE levels were also increased, it was unclear if the increase in the nuclear pool simply reflected a proportional increase that correlated with the total cellular apoE level, or if starvation stimulates apoE nuclear transport. Potentially relevant to the latter theory, starvation has been shown to promote the nuclear accumulation of proteins such as Hsp70 .
In the present study, we used apoE–GFP (green fluorescent protein)-expressing cells and biotinylation approaches to study potential nuclear trafficking of endogenous and exogenous apoE respectively. The results indicate that a small but detectable amount of endogenous apoE–GFP can be demonstrated in the nucleus and that nuclear apoE–GFP levels can be increased by serum starvation. Furthermore, exogenous biotinylated apoE can also be transported to the nucleus, but only if the apoE is presented to the cell as a lipoprotein complex. These findings may have implications for an intracellular function of apoE beyond that of lipoprotein transport.
MATERIALS AND METHODS
Cell culture media and additives were obtained from Invitrogen unless stated otherwise. The human cell lines SK-N-SH and foreskin fibroblasts (AG01518) were obtained from the A.T.C.C. (Manassas, VA, U.S.A.) and the Coriell Institute for Medical Research (Camden, NJ, U.S.A.) respectively. Cells were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FCS (fetal calf serum), 2 mM L-glutamine, penicillin (100 i.u./ml) and streptomycin (100 μg/ml) at 37 °C in humidified air containing 5% CO2. The CHO (Chinese-hamster ovary) apoE–GFP cell line was established as described previously  and cultured in Ham's F12 medium containing 10% (v/v) FCS, 2 mM L-glutamine, penicillin (100 i.u./ml), streptomycin (100 μg/ml) and geneticin (300 μg/ml). For starvation studies, the cells were initially grown in the normal Ham's F12 medium until reaching confluency, and then were switched to serum-free medium for 2 weeks, with a medium change every 3 days.
CHO apoE–GFP cells were cultured on coverslip-based 35 mm diameter Petri dishes until the cells reached approx. 80% confluency. They were then transferred to a live-cell imaging Nikon TE2000 inverted microscope fitted with an environmental chamber/CO2 incubator (Solent Scientific, Segensworth, U.K.) and Prior stage (Prior Scientific, Rockland, MA, U.S.A.). Cells were maintained at 37 °C and humidified 5% CO2, and GFP images were collected using a blue excitation filter block (part number 41001; Semrock, Rochester, NY, U.S.A.). Z-stacks were routinely collected at 1 μm spacing, and for time course studies intervals of 20–60 s were selected. Time course series were compiled using Image Pro-Plus v6.1 (Media Cybernetics, Bethesda, MD, U.S.A.) and Nearest Neighbour Deconvolution was applied to Z-stacks using the Sharp Stack function.
ApoE–GFP-expressing CHO cells were fixed using a combination of 4% (w/v) paraformaldehyde in PBS and methanol/acetone (1:1, v/v). Briefly, cells were treated with 4% (w/v) paraformaldehyde for 5 min at 4 °C, followed by a 10 min incubation at 22 °C. Cells were then rinsed in PBS and treated with methanol/acetone (1:1, v/v) for 6 min at −20 °C . Cells were then permeabilized with 1% (w/v) NP40 (Nonidet P40) (Sigma) and 10% (v/v) goat serum in PBS for 20 min at 22 °C, then treated with mouse monoclonal anti-[human Sm (Smith) antigen] Y12 antibody (1:400 dilution; Abcam, Sapphire Bioscience, Redfern, NSW, Australia) for 1 h at 22 °C. After rinsing in 1% (w/v) NP40, cells were incubated with Alexa Fluor® 568-conjugated goat anti-(mouse IgG) (1:100 dilution; Invitrogen) for 1 h at 22 °C. Samples were again rinsed three times with 1% (w/v) NP40 and PBS and then mounted in Vectashield (Vector Laboratories, Burlingame, CA, U.S.A.). Specimens were studied using a Nikon TE2000 microscope and a ×60 Plan-Apochromat oil immersion objective lens (1.45 numerical aperture; Nikon). Fluorescence was detected using Semrock filter sets and an excitation/emission filter block of 472/520 nm for GFP and 562/624 nm for Alexa Fluor® 568 staining.
Cells were rinsed with cold PBS and lysed in RIPA buffer [20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP40, 0.5% deoxycholate and 0.1% SDS] containing protease and phosphatase inhibitors. BCA (bicinchoninic acid) protein assays were performed on lysates and equal amounts of protein were resolved by SDS/PAGE (12% gels) and transferred on to nitrocellulose membranes at 100 V for 30 min. Membranes were blocked overnight at 4 °C in PBS containing 5% (w/v) non-fat dried skimmed milk and 0.1% Tween 20 and probed with rabbit polyclonal anti-apoE (1:1000 dilution; Dako), rabbit polyclonal anti-GFP (1:4000 dilution; Invitrogen), mouse monoclonal anti-(Sm antigen) (1;200 dilution; Abcam), rabbit polyclonal anti-nucleolin (1:500 dilution; Santa Cruz Biotechnology), mouse monoclonal anti-biotin (1:1000 dilution; Sigma) and rabbit polyclonal anti-β-actin (1:2000 dilution; Dako) antibodies. Anti-β-actin was used as a loading control. The membranes were washed three times in PBS containing 0.1% Tween 20 and then incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:2000 dilution; Dako) or rabbit anti-mouse (1:2000 dilution; Dako) secondary antibodies for 2 h. Signals were detected using ECL® (enhanced chemiluminescence) (Amersham Biosciences) and X-ray film.
Isolation of nuclei
Cell nuclei were isolated using the Nuclei EZ Prep nuclei isolation kit (Sigma) following the manufacturer's instructions as detailed previously . Briefly, cells were rinsed with ice-cold PBS, lysed with ice-cold Nuclei EZ lysis buffer and scraped up with a bladed cell scraper. The lysate was collected, vortexed briefly and centrifuged at 500 g for 5 min at 4 °C. The supernatant was removed and the nuclei resuspended in the lysis buffer and centrifuged as above. The pellet was resuspended in 200 μl of ice-cold Nuclei EZ storage buffer, a BCA protein assay was performed and equal amounts of nuclear protein were resolved by PAGE. Prior to PAGE and Western blotting, the nuclear preparations were treated with DNaseI for 1 h at 37 °C. It is established that β-actin is a component of mammalian chromatin-modifying complexes , and therefore blotting for nuclear β-actin was used to confirm equal protein loading of nuclear lysates.
Nuclear speckle isolation
Subnuclear structures known as interchromatin granule clusters or nuclear speckles were isolated from nuclei as described previously . Briefly, cell nuclei were resuspended in ice-cold TM5 buffer [10 mM Tris/HCl (pH 7.4) and 5 mM MgCl2 (pH 7.4)] containing protease inhibitors, incubated with 1% (v/v) Triton X-100 and 2 mM vanadium ribonucleoside complex for 5 min on ice and then centrifuged at 780 g for 5 min at 4 °C. The pellet was resuspended in TM5 buffer and treated with DNaseI for 1 h at 4 °C, with intermittent mixing. NaCl (0.5 M final concentration) was added, incubated on ice for 5 min and centrifuged at 770 g for 5 min at 4 °C. The pellet was resuspended in TM5 buffer containing 0.5 M NaCl and centrifuged as before, and this process was repeated prior to resuspension of the pellet in TM5 buffer containing 0.5 M NaCl and 5 mM dithiothreitol and incubation on ice for 5 min. The homogenate was then passed 10 times through a needle (27 gauge) and homogenized 20 times using a Dounce homogenizer. The homogenate was then mixed with TM5 buffer containing 0.2 M Cs2SO4 and centrifuged at 20800 g for 2 min at 4 °C. The supernatant was transferred to polycarbonate tubes (Beckman) and centrifuged at 60000 rev./min in a Beckman Optima TLX ultracentrifuge (TLA 100.3 rotor) for 1 h at 4 °C. The speckle pellet was resuspended in 30 μl TM5 buffer and stored at −20 °C.
Isolation and characterization of secreted apoE–GFP
ApoE–GFP-expressing CHO cells were grown in Ham's F12 medium containing serum and antibiotics as above. Once confluent, cells were washed twice with fresh Ham's F12 medium without serum or antibiotics and cultured in the same medium for a further 24 h. The CCM (cell-conditioned medium) containing secreted apoE–GFP was collected and centrifuged at 1000 g for 5 min at 22 °C to remove any cellular debris. The CCM was then concentrated using Centricon YM-50 membrane filters (Millipore) following the manufacturer's instructions. The concentrated medium was analysed by SDS/PAGE (12% gels) and gels were stained with Coomassie Brilliant Blue solution [0.1% Coomassie Brilliant Blue G250, 40%(v/v) methanol and 10% (v/v) acetic acid] for 2 h and destained with methanol/acetic acid/water (4:1:5, by vol.).
ApoE synthesis and biotinylation
Recombinant human apoE (E2, E3 and E4 isoforms) were prepared from Escherichia coli as described previously [26,27]. ApoE discs containing apoE, POPC (1-palmitoyl-2-oleoyl phosphatidylcholine) and cholesterol were prepared using the cholate dialysis method and characterized as described by Kim et al. . The apoE discs had an average diameter of 17.0 nm as judged by gel-filtration chromatography . The POPC/cholesterol/apolipoprotein molar ratio of the apoE discs was approx. 105:11:1. The disc size, apoE conformation and lipid composition resembled nascent apoE discs secreted from astrocytes . ApoE and apoE discs were biotinylated as described previously . Briefly, 0.2 mg of apoE and apoE disc protein were treated with 1.5 mg of EZ-Link Sulfo–NHS (N-hydroxysuccinimido)–Biotin (Pierce) in 1 ml PBS for 4 h at 4 °C and then dialysed using a Slide-A-Lyzer dialysis cassette [10 kDa MWCO (molecular mass cut-off); Pierce] in PBS overnight at 4 °C with four buffer changes. To study the nuclear trafficking of exogenous biotinylated apoE or apoE discs, we adapted a method described previously . SK-N-SH cells were treated with 20 μg/ml biotinylated lipid-free apoE or biotinylated apoE discs for up to 24 h, rinsed twice with fresh DMEM, and incubated for a further 2 h in fresh medium prior to a final rinse with PBS. Cells were then lysed directly or subjected to the nuclear isolation protocol described above. As a biotin-only control, cells were treated for 24 h with 250 μg/ml biotin, which is greatly in excess of the concentration of free biotin that would be predicted to remain after dialysis. This control condition was added to ensure that any biotinylated signal detected by Western blotting was not the result of non-specific labelling of cellular/nuclear proteins that was caused by traces of biotin remaining after the dialysis step.
Cholesterol efflux assay
Cellular cholesterol efflux was used as an index of apoE or apoE disc structural integrity as described previously . Briefly, SK-N-SH cells were labelled with 2 μCi/ml [3H]cholesterol (Amersham Biosciences) for 24 h, rinsed with PBS and incubated for 2 h in DMEM containing 0.1% BSA to allow equilibration of [3H]cholesterol in intracellular pools. The cells were then rinsed once in PBS and then incubated in serum-free DMEM containing 0.1% BSA with apoE3 (15 μg/ml) or apoE3 discs (15 μg/ml) for 24 h. Samples of the medium were collected at specific times and cleared of any cellular debris by centrifugation at 1000 g for 5 min at 22 °C. The cells were lysed with 0.1 M NaOH and the radioactivity in the medium samples and cell lysates was measured by scintillation counting. Cholesterol efflux to the medium was calculated as a percentage of total radioactivity in the cell lysates and medium.
Experiments were routinely performed in triplicate and repeated at least twice. Where indicated, results are means±S.E.M. and statistical significance was determined using the Student's t test, with P<0.05 considered significant.
Nuclear localization of apoE–GFP in CHO cells
Previous studies indicated that apoE can be trafficked to the nucleus [9,19,20]. We sought to confirm and extend these observations using CHO cells that had been stably transfected to express apoE–GFP (isoform E3) under the control of a CMV (cytomegalovirus) promoter . Live-cell microscopy indicated that there was strong expression of apoE–GFP in the majority of cells (Figure 1A), with apoE–GFP predominantly present in the cytosol, apparently within secretory vesicles and in association with the endoplasmic reticulum and Golgi compartments (Figure 1A). A small proportion of apoE–GFP was also detected within the nucleus. Using multiple Z-plane focus positions and live cells, it was clear that numerous speckle-like structures ∼0.3–1 μm in diameter were present throughout the nucleus (Figures 1A and 1B). With time course experiments and single cell analysis, nuclear and cytosolic pools could be clearly discriminated and apoE–GFP was also detected in association with cytoskeletal structures that, on the basis of previous work [32,33], are very likely to include microtubules (see Supplementary Figure 1 at http://www.BiochemJ.org/bj/409/bj4090701add.htm). Occasionally, small apoE–GFP cytosolic vesicles were observed to travel along the path of the apoE–GFP-positive cytoskeletal structures (see Supplementary Figure 1).
Confocal laser-scanning microscopy confirmed that apoE–GFP was present in the nucleus in a punctate pattern (Figure 1C). Starvation induced by serum withdrawal has been shown previously to induce growth arrest and increase fibroblast apoE expression in whole-cell lysates and nuclear extracts [9,11]. To assess whether apoE–GFP nuclear targeting was modulated by starvation, apoE–GFP-transfected CHO cells were initially grown in complete medium and then cultured in medium lacking serum for 2 weeks. As a control, cells grown only in complete medium were also prepared. As expected, serum starvation of the CHO apoE–GFP cell line resulted in growth arrest (results not shown), with live-cell microscopy indicating a dramatic change in the appearance of apoE–GFP in the cytosol which appeared to coalesce to form large intracytosolic structures (Figure 1D). The appearance of apoE–GFP in the nucleus was also altered under serum starvation, with a more diffuse signal detected in addition to the punctate appearance observed in cells cultured in the presence of serum (Figure 1D).
In order to determine whether the absolute levels of apoE–GFP were affected by serum starvation, cellular and nuclear proteins were isolated from the serum-starved and control cells, analysed by Western blotting and quantified using NIH (National Institutes of Health) ImageJ software (Figure 2). A 52 kDa protein corresponding to apoE–GFP was highly expressed in cell lysates, and protein levels were similar under the serum-starved and the control conditions (Figure 2). In contrast, nuclear apoE–GFP levels were increased with starvation (Figure 2).
The mechanism of apoE entry into the nucleus is unlikely to be through simple diffusion through the nuclear pores, as apoE–GFP at 52 kDa is probably too large to enter by this route . In addition, the increase in nuclear apoE–GFP localization under starvation conditions, which is not simply the result of increased expression levels, implies an active mechanism of nuclear accumulation.
Nuclear apoE–GFP is not associated with interchromatin granule clusters/nuclear speckles
The punctate appearance of nuclear apoE–GFP and the general absence of signal in the nucleolus (see enlarged insert, Figure 1D) led us to speculate that apoE may be associated with interchromatin granule clusters/nuclear speckles [25,35]. Nuclear speckles are subnuclear structures enriched in pre-messenger RNA splicing factors including snRNPs (small nuclear ribonucleoproteins). Using the Y12 antibody to detect Sm antigen as a marker for nuclear speckles , we observed very little co-localization of the speckle marker with apoE–GFP (Figure 3A). In additional experiments, the nuclear speckle fraction was isolated from CHO apoE–GFP nuclei by ultracentrifugation , and the nuclear and speckle proteins were analysed by Western blotting (Figure 3B). The speckle-specific protein, the Sm antigen, was detected in the nucleus and highly enriched in the speckle fraction as expected (Figure 3B). In contrast, apoE–GFP was clearly detected in the nuclear fraction, but present at only low levels in the speckle fraction and was, therefore, not enriched in nuclear speckles (Figure 3B). The nuclear and speckle fractions were also probed for nucleolin, which should not be present in nuclear speckles. As predicted, nucleolin was present in the nuclear fraction but not detected in the nuclear speckle fraction, indicating that the speckle fraction was free of contaminating proteins. These results indicate that despite the speckle-like distribution of apoE–GFP in the nucleus, interchromatin granule clusters/nuclear speckles are not the primary location of nuclear apoE–GFP.
Trafficking of exogenous apoE to the nucleus
To determine whether secreted apoE–GFP could be targeted to the nucleus, serum-free growth medium from CHO apoE–GFP cells was collected, concentrated and semi-purified using Centricon filters before being incubated with non-GFP-expressing recipient cells. Western blot analysis of the cell culture medium from CHO apoE–GFP cells indicated the presence of the expected 52 kDa apoE–GFP protein (Figure 4A). After filter concentration and purification, the 52 kDa apoE–GFP protein accounted for ∼70% of proteins detected on Coomassie-stained SDS/PAGE gels (Figure 4B). In addition, ∼90% of the apoE-positive material was present in the 52 kDa form, indicating that the secreted apoE–GFP remained intact and could be concentrated by approx. 30-fold (Figure 4B). The concentrated and purified apoE–GFP was then diluted in growth medium and incubated with non-apoE–GFP-expressing CHO cells or SK-N-SH neurons for up to 24 h. The cells were then rinsed and pulsed for 2 h with growth medium devoid of apoE–GFP, and the presence of apoE–GFP in cell lysates and isolated nuclear fractions was examined by Western blotting.
When the apoE–GFP protein concentrate was applied exogenously to control CHO cells, apoE–GFP was not detected in either cellular or nuclear fractions (Figure 5A). In similar experiments, apoE–GFP was also applied exogenously to SK-N-SH cells and, once again, the apoE–GFP protein was not detected (Figure 5B). Similar results were also obtained when cells were treated for only 1 h with apoE–GFP (results not shown). Since the structure of apoE–GFP is not identical to native apoE and it is not part of a lipoprotein complex, it is possible that apoE–GFP may not interact optimally with endocytic receptors. The lack of detection of apoE–GFP in association with control CHO cells and SK-N-SH neurons could therefore be the result of inefficient uptake of apoE–GFP. However, earlier studies have shown that apoE–GFP can be taken up by human fetal brain cell cultures by a process which was presumed to be receptor-mediated endocytosis . To address the lack of apoE–GFP signal in the SK-N-SH neurons, we conducted an additional control experiment to assess whether apoE expression could be up-regulated by serum starvation and if this was associated with an increase in nuclear apoE (i.e. to ensure the potential for nuclear trafficking of apoE exists in this cell type). The results from this experiment indicated that apoE was barely detectable by Western blot in SK-N-SH cells under standard culture conditions, but was induced with starvation, and this was associated with a parallel increase in nuclear apoE (Figure 5C). This result was very similar to observations made using human fibroblasts (Figure 5D), which were used as a positive control for the experimental system, and thus confirmed our previous results . We therefore conclude that under the experimental conditions described, apoE–GFP is possibly endocytosed and degraded before it has an opportunity to reach detectable levels in the nucleus or other intracellular compartments.
Analysis of biotin-labelled apoE
To avoid possible issues associated with the bulky GFP tag interfering with binding, internalization and degradation, we used biotin as an alternative molecule to label and track exogenous apoE (isoform E3). Biotin is a small molecule that covalently binds lysine residues (of which there are 12 in human apoE). Two forms of recombinant apoE were labelled with biotin: lipid-free apoE and apoE discs (containing phospholipids and cholesterol). The apoE discs resemble nascent brain lipoproteins and are known to play a role in the regulation of neuronal cholesterol transport . The biotinylated apoE preparations were extensively dialysed to remove unbound biotin and then analysed by Western blotting. The molecular mass of the biotinylated apoE protein increased from 34 kDa to 38 kDa, reflecting the addition of biotin bound to multiple lysine residues (Figure 6A). The structural integrity and associated function of lipid-free apoE and apoE discs was confirmed in cholesterol efflux assays, where both forms of apoE were able to stimulate cholesterol efflux from SK-N-SH neurons (Figure 6B), consistent with previous results . To test whether exogenously added apoE could be taken up by cells, the biotinylated lipid-free apoE and apoE discs were applied to SK-N-SH neurons. After a 24 h incubation with biotinylated apoE or apoE discs, followed by a 2 h chase with standard DMEM and washing with PBS, cell lysates were analysed by Western blotting. The apoE–biotin protein was detected only in cells that were treated with biotinylated apoE discs, not in untreated cells or in cells treated with lipid-free biotinylated apoE (Figure 6C). This demonstrates that exogenously added apoE can accumulate in cells and furthermore that the lipidated form appears to be either stabilized or at least partly trafficked to a non-destructive intracellular compartment.
Nuclear localization of biotinylated apoE
As we demonstrated that biotinylated apoE discs can accumulate in SK-N-SH neurons, additional experiments were conducted to assess whether biotinylated apoE could be detected in the nucleus. As described above, SK-N-SH cells were treated with biotinylated apoE discs, chased, washed, and cellular and nuclear proteins were analysed by Western blotting. Figure 7 indicates that biotinylated apoE was detected both in whole-cell lysates and in isolated nuclei. In the untreated control conditions, native apoE was below the limit of detection in these experiments and, as expected, biotinylated apoE was not present.
Because the apoE genotype is a determinant of apoE function and a risk factor for cardiovascular disease and Alzheimer's disease [1,5,38], we assessed the nuclear trafficking of biotinylated apoE discs containing each of the three common human apoE isoforms (E2, E3 and E4). SK-N-SH cells were treated with the three biotinylated apoE discs as described above, chased, washed, and cellular and nuclear proteins were analysed by Western blotting. All three apoE isoforms were equally detected in both whole-cell lysates and in isolated nuclei (Figure 7B).
Although the role of apoE as a secretory extracellular protein has been extensively studied, the possible functions of intracellular apoE are not well understood. Intracellular apoE regulates the routing of internalized lipoprotein remnants, and is involved in the assembly and secretion of VLDL (very-low-density lipoprotein) [39–41]. In addition, the endocytosis of apoE–lipoprotein complex initiates signal transduction [17,42]. Whether apoE targeting to the nucleus underscores yet another function of apoE remains unknown.
The exact pathway by which apoE, a protein normally destined for secretion, is able to reach and enter the nucleus remains unknown. Nucleocytoplasmic trafficking requires that proteins cross the nuclear membrane via the nuclear pore complexes. Small proteins (less than 40–50 kDa) can passively diffuse through nuclear pore complexes unassisted, whereas relatively large proteins (>50 kDa) require an active transport pathway largely mediated by importins [34,43]. Our results indicated that apoE–GFP at 52 kDa could be detected in the nucleus, which suggested that an active transport pathway may be involved, rather than a diffusion-based mechanism.
In humans, apoE exists as a glycoprotein containing a single O-linked oligosaccharide at Thr194 . It is not known if apoE glycosylation can modulate its trafficking, but it is possible that the hydrophilic glycan moiety may have some impact; for example, during transmembrane movement. It is therefore possible that the recombinant human apoE used in some of our experiments may behave differently from native glycosylated apoE.
Nucleocytoplasmic trafficking usually requires the presence of NLSs (nuclear localization sequences). NLSs are short stretches of basic amino acids existing as a single cluster (monopartite) or as two clusters separated by a spacer region (bipartite) . ApoE does not appear to contain the classical monopartite or bipartite NLS, however it does contain two polybasic domains that fulfil the criterion for potential ‘weak’ NLSs, as defined by the presence of four to five basic residues in a hexapeptide [46,47]. Whether these weak NLSs are functional remains to be determined. It should also be noted that specific proteins may translocate to the nucleus without having an apparent NLS. One example is the growth factor midkine, which is involved in neuronal survival and differentiation . Midkine binds to LRP (LDLr-related protein) on the cell surface and, once internalized, utilizes nucleolin as a nuclear targeting chaperone . Our results indicate that extracellular apoE can be trafficked to the nucleus when presented to the cell in the form of a lipidated disc, suggesting a pathway that shares features in common with the midkine nuclear-targeting pathway could therefore be involved. As noted previously, apoE binding to nucleolin or LRP and heparan sulfate proteoglycans on the cell surface may result in apoE being trafficked to the nucleus in a manner analogous to midkine, fibroblast growth factor and other heparin-binding proteins (see  and references therein).
Previous work indicated that small amounts of apoE can be detected in the cytosol in association with microtubules . Our observations derived from the living CHO apoE–GFP cell line (see Supplementary Figure 1) concur with this previously published work. Interestingly, certain microtubule-associated proteins can be transported to the nucleus via a dynein-dependent pathway [49–51]. It is possible therefore that apoE–GFP may be trafficked to the nucleus via microtubules. The alternative possibility, that apoE–GFP may be initially secreted from CHO apoE–GFP cells and then interact with cell-surface molecules that facilitate or mediate its transport to the nucleus, seems unlikely, as exogenously added apoE–GFP did not result in the appearance of apoE–GFP in the nucleus.
Our current results also indicate that nuclear apoE–GFP levels were increased under serum starvation conditions. Previous work has shown that nucleocytoplasmic trafficking of proteins is dependent on certain stress conditions. Interestingly, starvation can induce nucleocytoplasmic trafficking and promote accumulation of specific proteins in the nucleus [21,52]. It appears that the accumulation of specific proteins in the nucleus may be an adaptive survival response to changes in the extracellular environment . It remains to be determined whether targeting of apoE to the nucleus represents a generalized cell survival response.
In conclusion, we have shown that endogenous apoE–GFP is detected in the nucleus and that the level of nuclear apoE–GFP is increased with serum starvation. Furthermore, exogenous biotinylated apoE can also be transported to the nucleus if apoE is presented to the cell as a lipoprotein complex. These studies provide new evidence that apoE may be targeted to the nucleus and shed light on factors that regulate this process.
This work was support by the Australian Research Council (Grant No. DP0557295). B. G. is supported by an R. D. Wright Fellowship from the Australian National Health and Medical Research Council. We are grateful to Professor Karl Weisgraber (Gladstone Institute of Neurological Disease, University of California San Fransisco, San Fransisco, CA, U.S.A.) for provision of human apoE cDNA.
Abbreviations: ApoE, apolipoprotein E; BCA, bicinchoninic acid; CCM, cell-conditioned medium; CHO, Chinese-hamster ovary; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GFP, green fluorescent protein; LDLr, low-density lipoprotein receptor; LRP, LDLr-related protein; NLS, nuclear localization sequence; NP40, Nonidet P40; POPC, 1-palmitoyl-2-oleoyl phosphatidylcholine; Sm, Smith
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