Post-translational Regulation of Macrophage Apoprotein E Production*

We have transfected the murine macrophage cell line, 5774, which does not express its endogenous apoE gene, with a constitutively expressed human apoE cDNA in order to study post-transcriptional and post-translational control of apoE production in macro- phages. Loading cells with cholesterol using preincubations in acetylated low density lipoprotein, previ- ously shown to enhance macrophage apoE gene transcription and apoE synthesis, did not increase apoE synthesis or secretion in constitutively expressing transfected cells, suggesting that sterol control of mac- rophage apoE production occurs predominantly at a transcriptional locus. However, incubation in human high density lipoprotein (HDL3) stimulated apoE secre- tion and appeared to inhibit degradation of newly synthesized apoE. This effect could be entirely reproduced by incubation with phosphatidylcholine vesicles which increased apoE accumulation in the medium by 2-6-fold. Pulse-chase experiments indicated that the effect of HDLs or phospholipid vesicles was very rapid (oc-curring within 15 min) and was independent of changes in apoE synthesis. Furthermore, the increased apoE which accumulated in the medium in the presence of phospholipid vesicles or HDL3 was not due to altered rates of reuptake of labeled apoE since this difference was completely preserved in the absence of extracel- lular calcium. These results indicate that alteration of sterol content does not regulate within the but to participate in HDL3-mediated net apoE cells

latter cell type, expression of apoE mRNA is regulated independently of expression of lipoprotein lipase but is positively correlated with differentiation from preadipocytes to adipocytes and with cellular lipid content, particularly free cholesterol content (5). In rat ovarian granulosa cells, apoE production has been shown to be responsive to stimulation by follicle-stimulating hormone, cAMP agonists, and phorbol ester (3,4). Interestingly in this cell type, the quantitative response in the accumulation of secreted apoE protein substantially exceeds the increment in apoE mRNA abundance in response to both cAMP and phorbol esters. This observation led the authors to hypothesize a potential post-transcriptional locus of control for apoE production in this model system. In macrophage-type cells, apoE production is modulated as a function of differentiation of monocytes to a macrophage-like phenotype (6,7), exposure to bacterial endotoxin or other activating agents (8,9), and cellular sterol balance (1,2,(10)(11)(12). In particular, macrophage-free cholesterol has been shown to positively regulate apoE gene expression (10,11).
ApoE gene transcription rate is increased 8-fold after cholesterol loading of macrophages (11); apoE mRNA levels are decreased by induction of negative cholesterol balance in cholesterol-loaded macrophages; and apoE and low density lipoprotein receptor mRNA abundance vary inversely over an identical range of macrophage cholesterol content (11,12). All of the above observations emphasize the importance of apoE gene response to perturbations of macrophage cholesterol homeostasis but do not address potential post-transcriptional loci of sterol control. Such loci have been well documented for other sterol-responsive pathways (13) (for example hydroxymethylglutaryl-CoA reductase) and have been suggested for apoE in the steroidogenic cell model (3,4) (see above) and by recent data in macrophages suggesting that HDL3 interaction with a plasma membrane receptor could modulate apoE secretion (14). In this series of studies, we have further examined the question of potential post-transcriptional control of apoE in macrophages using the mouse 5774 cell model system (which produces no endogenous apoE) transfected with a constitutively expressed human apoE cDNA. Such a model precludes regulation at the level of gene transcription or subsequent processing of nascent apoE transcripts. Our results suggest that alterations in macrophage sterol content do not regulate macrophage apoE production at translational or post-translational loci (ie. sterol regulation is transcriptional) but that HDL, can stimulate apoE secretion independent of changes in gene transcription. The effect of HDL3 is independent of net cholesterol efflux and can be reproduced by incubations in phospholipid vesicles suggesting that neither HDL3 apoproteins nor interaction with an apoprotein receptor are necessary for post-transcriptional regulation of apoE production by macrophages. Cell Culture"J774 macrophages were transfected with a constitutively expressing human apoE cDNA. A cDNA expression vector containing the human metallothionine IIA promoter and SV40 16s splice junction and polyadenylation signal was prepared. The 840base pair HindIII-BamHI fragment of pHSI (15) (obtained from Michael Karin, University of California, San Diego) containing the human metallothionine IIa promoter was ligated to the XhoI-PstI fragment of pL1 (16) containing the 16 S splice junction and subcloned into the HindIII-PstI site of pUC19. The HindIII-KpnI fragment was then subcloned into the HindIII-KpnI-digested pcDVl vector (16). The AatII-HinfI fragment of the human apoE cDNA pE368 (17) was subcloned into the unique XbaI site downstream from the promoter after generating blunt ends and the addition of XbaI linkers to the apoE fragment.
Stably transfected 5774 cells were prepared by cotransfection with the apoE cDNA expression vector and pSV2-neo by calcium phosphate coprecipitation (18). Five X lo6 cells were treated with 25 pg of linearized apoE expression vector and 25 ng of linearized pSV2-neo for 4 h in the presence of 10 mM ammonium chloride. Transfected cells were selected with 400 pg/ml G418 and grown to confluency, and cells derived from a single G418-resistant clone were used for these studies. Cells transfected with pSV,-neo without apoE did not synthesize or secrete detectable apoE.
Cultures were maintained at 37 "C in a humidified incubator (5% COP) in 75-cm2 flasks containing 12 ml of growth medium consisting of Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, and 200 pg/ml G418. G418 was removed from the growth medium 2 weeks prior to initiation of experiments. Cells were removed from the culture flask by scraping.
Lipoproteins and Phospholipid Vesicles-Human HDL3 (d = 1.125-1.210 g/ml) was prepared by ultracentrifugation in KBr as previously described (10). Phosphatidylcholine vesicles were prepared as described by Batzri and Korn (19). Vesicles were dialyzed overnight against methionine-free DMEM plus 1% penicillin-streptomycin and filtered through a 0.45-pm membrane prior to use. r5S]Methionine Labeling of Cells-3 X lo6 cells were plated into 35-mm wells and grown for 48 h. All pulse media, chase media, and wash media used in the following procedures were warmed to 37 "C before use. Cells were washed twice between medium changes with methionine-free DMEM + 1% penicillin-streptomycin. All media were added to cells at a volume of 1 ml/well. For most experiments, cells were labeled by incubation with pulse medium either 1) for 2 h followed by a 6-h incubation with chase medium (with or without 1 mg/ml vesicles or 400 rg/ml HDL, added to the chase medium only or 2) for 6 h (with or without 1 mg/ml vesicles or 400 pg/ml HDL3) with no chase period.
Pulse medium consisted of 100 pCi/ml [35S]methionine and 10 p~ unlabeled methionine in methionine-free DMEM plus 1% penicillinstreptomycin. For experiments involving a pulse period shorter than 2 h, the concentration of unlabeled methionine was reduced to 2.375 p~. In all cases, chase medium was 500 p~ unlabeled methionine in DMEM plus 1% penicillin-streptomycin.
At the end of the incubation period, the medium was removed to sterile microcentrifuge tubes, aprotinin (100 kallikrein-inactivating units) and phenylmethylsulfonyl fluoride (0.1 mM) were added, and the medium was spun in a microcentrifuge at 4 "C for 15 min to remove any floating cells. Supernatant (900 pl) was stored at -20 "C. The cell layers were washed twice with phosphate-buffered saline containing aprotinin (100 kallikrein-inactivating units/ml) and phenylmethylsulfonyl fluoride (0.1 mM). The cells were frozen on the culture plate at -20 "C until cell lysis was performed.
Lysis of 35S-Labeled Cells-To lyse cells, 100 pl of 2% SDS was added to each well, and the cells were heated to 95 "C for 3 min. 900 p1 of cell lysis buffer (10 mM Na2HP04, 15 mM NaC1, 10 mM methionine, 1% Triton X-100, 1% deoxycholate) was added to each well, and the cells were incubated at 37 "C for 2 h. To decrease viscosity, the cell lysate was sheared twice through a 25-gauge needle. The lysate was spun in a microcentrifuge for 15 min. 850 p1 of cell lysate was stored at -20 "C until immunoprecipitation was performed.
Immunoprecipitation of 35S-Labeled Cell Lysates and Mediu-Antiserum against purified human apoE was prepared in New Zealand White rabbits as previously described (10). To determine incorporation into protein in cell lysate and media, trichloroacetic acid precipitation was done on 25 pl of each sample. In most cases, 4 X lo5 trichloroacetic acid-precipitable counts/min were immunoprecipitated from each medium sample, and 4 X lo6 trichloroacetic acidprecipitable counts/min were immunoprecipitated from each cell lysate sample. The required amount of each sample was aliquoted into sterile microcentrifuge tubes, and the volume of each sample was brought to 0.4 ml with sterile double distilled H20. 50 pl of 10 X PIA + methionine (100 mM Na2HP0,, 150 mM NaCl, 100 mM methionine, pH 7.4), 50 p1 of 10% Triton X-lOO/deoxycholate, and 10 pl of 10% SDS were added to each medium sample. Samples were precleared by the addition of 40 pl of Immunoprecipitin followed by incubation at room temperature on a rotator for 4 h and then spun in a microcentrifuge for 5 min. Supernatants were then transferred to fresh microcentrifuge tubes. For cell lysate samples, an incubation with nonimmune serum was required to reduce nonspecific binding to the apoE antiserum; therefore, 15 pl/tube of non-immune serum was added to the samples, and they were incubated on a rotator at 4 "C for 18 h prior to the addition of Immunoprecipitin.
After preclearing, 15 pl per tube of apoE antiserum was added. Samples were incubated at 4 "C on a rotator for 18 h. 40 pl of Immunoprecipitin was added to each sample, and incubation was continued at room temperature on a rotator for 4 h. Tubes were spun in a microcentrifuge for 5 min, and the supernatants were discarded. Pellets were resuspended in 1 ml of IP buffer (10 mM Na2HP04, pH 7.4, 15 mM NaCl, 10 mM methionine, 1% Triton X-100, 1% deoxycholate, 0.2% SDS) and spun in a microcentrifuge for 5 min. Supernatants were discarded and pellets were washed 3 more times with 1 ml of IP buffer. After the final wash, pellets were resuspended in 35 pl of SDS-polyacrylamide gel electrophoresis sample buffer + Pmercaptoethanol(62.5 mM Tris, pH 6.8,2% SDS, 5% P-mercaptoethanol) and heated at 95 "C for 5 min. Tubes were spun in a microcentrifuge for 5 min, 25 pl of supernatant/sample was transferred to fresh microcentrifuge tubes, and 2.78 pl of density solution (6.25 mM Tris, pH 6.8, 7.5% glycerol, 0.0075% bromphenol blue) was added. Samples were run on SDS-polyacrylamide gel electrophoresis with 3% stacking gel/lO% resolving gel.
After staining with Coomassie Blue, gels were prepared for fluorography with sodium salicylate according to Chamberlain (20). After drying, gels were exposed to Kodak X-Omat AR x-ray film with intensifying screens for 4-7 days. Flourographs were used to mark the position of the apoE bands on the dried gel. The dried gel piece from each sample was cut out and placed in a glass 20-ml scintillation vial, and the gel was rehydrated with 3% glycerol. The backing paper and glycerol solution were removed, and 0.5 ml of 30% HZ02 was added to each sample. The vials were capped tightly and heated at 60 "C for 48-72 h until the gel pieces dissolved. 10 ml of Biofluor scintillant was added, and the samples were counted against an external standard so that counting efficiency could be used to calculate disintegrations/min incorporated into apoE for each sample. In some cases, fluorographs were scanned using an LKB laser densitometer.
Other Assays-Cellular free and total cholesterol (after saponification) were measured in hexane/isopropyl alcohol extracts and analyzed by gas-liquid chromatography as previously described (10,11). Cholesterol ester was taken as the difference between total and free cholesterol. Cellular protein was measured in NaOH extracts by the method of Lowry using bovine serum albumin as standard (21).

RESULTS
In mouse peritoneal macrophages, human monocyte macrophages, and the THPl macrophage cell line, incubations in ALDL enhance apoE synthesis and secretion (1,2,10-12,221. We have previously shown that increased gene transcription contributes to this increase (11). In order to study potential post-transcriptional loci of sterol control, we utilized a transfected 5774 cell with an apoE cDNA expression vector containing the human metallothionine IIA promoter. Cells were pulsed with labeled methionine for 2 or 6 h and media and cell lysates were then analyzed for radiolabeled apoE content. As shown in Fig. 1, preincubations in ALDL did not enhance It has been previously suggested that HDL3 can induce apoE secretion in mouse peritoneal macrophages and that this effect was post-translational and dependent on interaction between HDL3 and a plasma membrane HDL3 receptor (14). Fig. 2 shows that addition of HDL3 (400 pg/ml) to the 5774 cells during the 6-h labeling period approximately doubles the amount of apoE secreted into the culture medium. HDL3 incubation with many cell types, including mouse peritoneal macrophages, produces net cholesterol efflux (10, 11, 23, 24). This effect can be detected either by a fall in total cholesterol content or, more sensitively, by an increase in endogenous cellular cholesterol synthesis. 5774 cells, however, unlike mouse peritoneal macrophages, do not respond to HDL3 with net cholesterol efflux (23). This "defect" in efflux has been well studied by Glick and colleagues, who have shown that HDL3 can promote net cholesterol efflux from 5774 cells only in the presence of CAMP, which also stimulated cholesterol ester hydrolysis (25). In Fig. 3 Fig. 5 show total labeled apoE secreted into the media over the chase period indicated; however, the results are essentially similar when the amount of apoE secreted is expressed on the basis of total labeled protein secreted. In Table I

TABLE I
The effect of phospholipid vesicles on apoE secretion expressed as a function of total labeled protein secreted 5774 cells were plated as described in the legend to Fig. 1 and after 48 h were pulse-labeled for 30 min followed by the indicated periods of chase. The disintegrations/min of total secreted apoE from each cell culture medium were divided by total secreted disintegrations/ min in trichloroacetic acid-precipitable protein and compared in the absence or presence of vesicles which were added at the start of the chase period. Values shown are based on comparisons of quadruplicate wells for each condition. Because the cell model utilized in these studies precludes transcriptional loci of regulation, it is likely that the differences observed in the experiments described above are due to differences in the cellular fate and/or stability of newly synthesized apoprotein E (ie. post-translational regulation). This issue is further examined in data presented in Table I1 which utilizes phospholipid vesicles to enhance apoE production. Immediately after the 30-min pulse time (zero time chase period) total counts (shown on the right) and cell lysate counts (shown on the left) are essentially the same, indicating that a neligible amount of labeled apoE was secreted by this time point. By 15 min into the chase period, there is a fall in the number of lysate apoE counts which is larger in the presence of phospholipid vesicles, but total apoE counts remain the same in the presence or absence of vesicles. The same changes are observed at the 30-min time point, that is, total counts remained the same; however, lysate counts again fall more substantially in vesicle-incubated cells than in control cells. By 90 min, however, the results are quite different. Lysate counts at this time period are identical in vesicle-treated and control cells and show a substantial fall in both circumstances. However, in the presence of vesicles, total counts have remained essentially stable between 30 and 90 min indicating that the large fall in cell lysate counts between 30 and 90 min was due to net secretion of labeled apoE. On the other hand, in control cells total apoE counts fall substantially between 30 and 90 min indicating net degradation of labeled apoE. These data then indicate that the incubation with phospholipid vesicles increases the secretion and enhances the stability of newly synthesized apoE. Macrophages have high affinity sites which bind and internalize apopoprotein E, and the ability of apoE to be recognized by these sites may be influenced by its secondary structure which in turn may be influenced by its association with lipid. It is therefore conceivable that the effects detected above are due to the ability of HDL3 or phospholipid vesicles to inhibit rapid reuptake of apoE and thereby perserve it against intracellular degradation after it has been secreted. To address this issue, cells were labeled for 30 min with high specific activity methionine and chased in the absence of calcium. High affinity apoE uptake in macrophages is absolutely dependent on calcium (26, 27), and therefore this protocol should eliminate observed differences if they were due to rapid reuptake and degradation of newly secreted apoE in control cells. To eliminate calcium from the chase medium, we employed two separate approaches. In one set of experiments, cells were chased in the presence of DMEM containing EGTA at a concentration adequate to chelate total medium calcium and magnesium. In the second experiment, cells were chased in the presence of calcium-and magnesium-free Hanks' balanced salt solution. The results are shown in Table 111. In the presence of EGTA, phospholipid vesicles and HDL3 produce a 5.9-and 9.1-fold increase in apoE secretion, respectively, during a 15-min chase. In the presence of Hanks' balanced

TABLE I11
The effect of removing extracellular calcium on HDL3 and phospholipid enhancement of apoE accumulation in the medium Cells were prepared and pulse-labeled for 30 min as described in the legend to salt solution the increases are 4.5-and 6.9-fold, respectively, during a 30-min chase. The data indicate that the apoE degraded in control cells (i.e. in the absence of phospholipid vesicles or HDL3) was not degraded as a result of secretion and subsequent reuptake and degradation in secondary lysomsomes but was, most likely, degraded prior to its secretion from cells.

DISCUSSION
The regulation of apoE production by macrophages has been widely studied. Differentiation state, exposure to endotoxin, and cellular cholesterol balance have all been shown to alter the amount of apoE secreted by macrophages (1,2,(6)(7)(8)(9)(10)(11)(12). The stimulatory effect of cholesterol loading, in particular, has been studied in mouse peritoneal macrophages, primary cultures of human monocyte-derived macrophages, and in the human monocyte-macrophage cell line THPl (1,2,(10)(11)(12)22). The measurement of apoE mRNA abundance and apoE gene nuclear run-off transcription in human and mouse macrophages has given clear evidence that the apoE gene transcription rate contributes to increased apoE secretion in sterol-loaded cells (11). The experiment shown in Fig. 1 was designed to address the importance of post-transcriptional control of macrophage apoE secretion in the presence of an expanded cellular sterol pool. There are well studied precedents for such regulatory loci in controlling the expression of other sterol-responsive proteins (13) (e.g. hydroxymethylglutaryl-CoA reductase) as well as other apolipoproteins (28,29) (e.g. apoB-100). As shown in Fig. 1,5774 macrophages, transfected to constitutively express a human apoE cDNA, do not synthesize or secrete more apoE after a 48-h incubation in ALDL in spite of a substantial increment in cellular cholesterol content. These results strongly suggest, therefore, that regulation of apoE gene transcription is the primary locus for sterol modulation of macrophage apoE secretion.
The data in Figs. 2-5, however, indicate that like hepatocyte apoB secretion (28,29), the amount of apoE secreted by macrophage is, in fact, subject to post-transcriptional and post-translational control. Incubation with human HDL3 substantially increases the amount of apoE secreted by cells transfected to constitutively express a heterologous apoE cDNA (Fig. 2). This result is consistent with Dory's observations in mouse peritoneal macrophages expressing their native apoE gene and protein (14). The effect of HDL3 on apoE secretion can be definitively separated from its effect on net cholesterol efflux in 5774 cells, since this cell line does not respond to HDL3 with net cholesterol efflux. Furthermore, the addition of CAMP which activates cholesterol flux from 5774 cells to HDL, does not further augment the apoE response to HDL,. However, this experimental result does not eliminate the possibility that the addition of phospholipid vesicles or HDL3 could lead to some rearrangement of cholesterol distributed in subcellular pools and thereby influence apoE production.
In studies using mouse peritoneal macrophages it has been suggested that HDL3 must interact with a macrophage plasma membrane receptor in order to modulate apoE secretion (14). This interaction, and a primary role for HDL3 apoproteins in this interaction, was suggested by the observation that tetranitromethane modification of HDL3 apoproteins abolished HDL3 stimulation of macrophage apoE secretion. In our system, the effect of HDL3 on apoE secretion could be entirely reproduced by incubation with phosphatidylcholine vesicles. While these observations do not establish that HDL3 effects are mediated by its phospholipid content, they demonstrate that the phospholipid component of HDL3 could completely account for the effect of HDL, on macrophage apoE secretion in our cell model. Because the cell model we have utilized constitutively expresses the human apoE cDNA, the changes noted in apoE protein in Figs. 2-4 must be due to changes in apoE synthesis (i.e. translation rates), apoE stability, or apoE secretion. The data from the pulse-chase experiments in Fig. 5 indicate that changes in apoprotein E translation rates do not account for our observations. In these experiments, HDL3 or phospholipid vesicles are added at the time the labeled methionine pulse is completed, and the decay of labeled apoE is followed for variable chase periods. As can be seen from the data in Table  11, there is a substantial fall in the amount of labeled apoE detectable in cell lysates between 30 and 90 min of chase in both control cells and in cells incubated with phospholipid vesicles. However, as can be seen from the preservation of total apoE counts over this same period in the presence of vesicles, the fall in lysate apoE is due to net degradation of labeled apoE in control cells, while it is due to net apoE secretion in cultures incubated with vesicles. Therefore, the addition of phospholipid vesicles at the beginning of the chase period preserves a substantial portion of the apoE synthesized during the prior 30 min from degradation. Whether the vesicles inhibit apoE degradation as a secondary effect of enhancing secretion ( e g . by promoting translocation from a pool where it is susceptible to degradation) or act primarily by inhibiting degradation will require additional studies. The direction of such investigations will need to take account of several important conclusions one can draw from the data in Fig. 5 and Tables I and 111.
First, it can be seen (Fig. 5) that the response to HDL3 or phospholipid vesicles is extremely rapid, occurring within 15 min of their addition. Second, the effect of phospholipid vesicles on apoE secretion does not reflect a generalized effect on macrophage protein secretion (Table I), and third, the effect of HDL, or phospholipid vesicles on apoE accumulation in the medium is not due to altered high affinity reuptake of secreted apoE since the effect is preserved even when high affinity degradation of apoE is abolished (26,27) by removing extracellular calcium (Table 111).
Human HDL3 is a complex particle made up of several species of apoproteins, cholesterol, and phospholipid. Dissecting the mechanism by which HDL, produces its effects on macrophage apoE secretion and degradation could be correspondingly complex. It could be expected that mechanistic dissection of the effect of single species phospholipid vesicles would be more straightforward. These vesicles may fuse with the plasma membrane of cells and thereby direct intracellular apoE to this location where secretion may be favored over intracellular degradation. Phosphatidylcholine vesicles have a reasonably high affinity for apoE as demonstrated by their ability to acquire apoE from @-very low density lipoprotein when they are co-incubated in vitro (30). Alternatively, phospholipid vesicles could be internalized and become associated with intracellular apoE and thereby reduce its susceptibility to degradation either directly or by altering its subcellular localization. Interestingly, in cryothin sections of hepatocytes, apoE can be detected by immunogold labeling in peroxisomes and in areas between multivesicular bodies and bile canaliculi (31). These observations suggest that apoE may function as an intracellular as well as a secreted protein. It is therefore possible that the presence of extracellular HDL3 or phospholipid modulates the distribution of intracellular apoE between a pool with a primarily intracellular function (and therefore destined for eventual intracellular degradation) and a pool targeted for eventual secretion.
Modulating the degradation of newly synthesized protein also appears to be important for determining the eventual secretion rate of apoB from hepatocytes. In HepG2 cells, up to 60% of newly synthesized apoB-100 is degraded (32). This degradation appears to occur in a pre-Golgi compartment and is insensitive to inhibition by chloroquine, leupeptin, pepstatin, or chymostatin, i.e. this degradation appears to occur in a non-lysosomal compartment closely related to the endoplasmic reticulum (29). The fraction of apoB-100 which is degraded intracellularly by HepG2 cells can be altered by the addition of exogenous oleate, which inhibits degradation and enhances secretion, or by insulin, which enhances intracellular degradation (28,33). The mechanism for the regulatory effects of oleate and insulin is unknown, but recently a great deal of information has become available regarding the details of the intracellular processing of apoB in hepatocytes (34-36). Much less is known about the intracellular processing and secretion of apoE in macrophages beyond the basic observation that apoE is secreted from macrophages in association with lipid, primarily phospholipid (1,37). It is now clear, however, that a more careful dissection of this process will be required in order to examine the mechanisms and implications of the multiple loci of control for macrophage apoE production.